Genetic, biochemical and neuropathological studies support the idea that cerebral elevation and accumulation of amyloid beta-peptide (Abeta) is an early and necessary step in AD pathogenesis. Abeta is generated from the amyloid beta-protein precursor (APP) by two proteolytic cleavages mediated by beta- and gamma-secretases. Mutations in the APP and presenilin genes cause rare early-onset forms of familial Alzheimer's disease (FAD), which shares many pathological features with the more common sporadic AD. Although FAD-associated genes (e.g. PS and APP) are expressed throughout the brain, early and most severe AD-associated neurodegenerative changes manifest in selective populations of neurons, e.g. neurons in entorhinal cortex and basal forebrain. Thus, additional cellular factors may contribute to selective neuronal vulnerability in AD. The hippocampus, a vital brain region for memory function, consists of multiple sub-regions each containing a unique population of neurons. The various subregions of hippocampus seem to be differentially affected in AD: for instance, entorhinal cortex is the most severely affected area, while dentate gyrus is relatively spared. To examine the underlying molecular mechanisms of selective vulnerability associated with common sporadic AD, we have performed carefully controlled microarray analyses to identify genes that are selectively up- or down-regulated in AD entorhinal cortex relative to dentate gyrus (in the same patient). Our microarray analyses revealed that VPS35p, a putative endosome/TGN trafficking molecule, is selectively upregulated in AD entorhinal cortex. Parallel analyses revealed that suppression of endogenous VPS35p using RNA interference (RNAi) in APP-expressing cells selectively lowers AP generation, suggesting that VPS35p is a modulatory factor in Abeta biogenesis. In light of our findings, the overall goal of this proposal is to perform molecular and functional characterization of VPS35p to delineate the role of this protein in APP processing and selective cellular dysfunction of entorhinal cortex in sporadic AD. The successful completion of this project will help identify cellular pathways that are directly involved in Abeta biogenesis and selective neuronal degeneration in AD entorhinal cortex, and may lead to development of a clinically valuable assay system for novel therapeutic reagents.